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Key Event Relationship Overview

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Description of Relationship

Upstream Event Downstream Event/Outcome
Induced Mutations in Critical Genes, Increased Proliferation/Clonal Expansion of Mutant Cells (Pre-Neoplastic Lesions/Altered Hepatic Foci (AHF)), Increased

AOPs Referencing Relationship

AOP Name Type of Relationship Weight of Evidence Quantitative Understanding

Taxonomic Applicability

Name Scientific Name Evidence Links

Taxonomic Applicability

Rats, Mice, Woodchucks, Humans, Monkeys, Birds, Trout, Tree shrews

This KER has not been directly observed, but there is indirect evidence in rats (Dycaico et al., 1996) and in neonatal mice (Chen et al., 2010). DNA repair and replication systems are conserved across eukaryotic species.

How Does This Key Event Relationship Work

There is no direct evidence addressing AFB1 induced critical gene mutations and the subsequent induction of AHF. However, in general the progression from chemical exposure to pre-neoplastic lesions appears to include six general mechanisms: 1) decrease in the activity of the Keap1/Nrf2/ARE pathway; 2) decrease in p53 function leading to increased survival in the presence of genomic instability; 3) changes in the tumor microenvironment within cells; 4) alterations in apoptosis; 5) changes in Wnt/b-catenin signaling, and 6) gene expression changes that may be related to cancer promotion/progression (Caballero et al., 2004; Ma and He, 2012; Gross-Steinmeyer and Eaton, 2012; Liby et al., 2008; Yates and Kensler, 2007; Liby and Sporn, 2012; Ikeda et al., 2004; Harida et al., 2004; Kwak et al, 2001; Honda et al., 2011; Shelton and Jaiswal, 2013; Johnson et al., 2014; Fabregat, 2009). In addition to these six possible mechanisms, epigenetic changes in DNA methylation may occur and may be related to the progression of cancer (Zhang et al., 2003, 2006, 2012; Wu et al, 2013; Pogribney et al., 2011).

Weight of Evidence

Biological Plausibility

AFB1 induces G:C to T:A transversions. The third nucleotide in codon 249 of the p53 gene is a guanine and is a preferred binding target for AFB1 metabolites. In humans, some evidence exists that this codon is a mutational hot spot in liver cancers in people exposed to AFB1. (Puisieux et al., 1991). Although there is a large literature describing mutations in this location in humans and the corresponding p53 codon 247 in rodents, whether these mutations by themselves actually lead to tumors is unknown. (Lee et al., 1998) In vitro data collected from two human cell lines suggest that doses of AFB1 sufficient to induce DNA damage also induce an incomplete cell cycle checkpoint response, likely involving p53; this suggests a potential mechanism that might ultimately lead to tumor formation in vivo.(Gursoy-Yuzugullu et al., 2011). In rats, Ki-ras codon 12 mutations observed after AFB1 dosing may also be associated with hyperplastic foci to a greater extent than in hepatocellular carcinomas (HCCs), suggesting some change in the mutational spectrum during tumor development. (Tokusashi et al., 1994)

In general, the exact origin of cells that form altered hepatic foci (AHF) is not known with certainty. (Sell, 1993, 2002; Sell and Leffert, 2008). Four types of cells in the liver are thought to be capable of becoming tumor cells: 1) mature hepatocytes, 2) bile duct progenitor cells, 3) ductular bipolar progenitor cells, and 4) periductular stem cells. Pre-neoplastic foci and HCCs likely form from the last two cell types.[1] In rats, pre-neoplastic foci expressing the placental form of GST appear to be descended from hepatocytes. (Bralet et al., 2002; Avril et al., 2004; Gournay et al., 2002).

However, Sell (2008) has criticized the hypothesis the HCC arise by de-differentiation of hepatocytes and suggested the cellular origin of HCC may be liver stem cells undergoing an arrest of maturation. Trosko (2014) has considered de-differentiation in light of one of the emerging “Hallmarks of Cancer”—alterations of cellular metabolism—and argues that the likelihood of cancer arising through de-differentiation is vanishingly small. (Hanahan and Weinberg, 2011; Trosko and Kang, 2012; Trosko, 2013, 2014). The questions of the origin of the progenitor cells that replace hepatocytes after liver injury and the origin of liver cancer stem cells are the subjects of intense research. (Gong et al., 2013; Jia et al., 2013; Miyajima et al., 2014; Rehman et al., 2014; Shin and Kaestner, 2014; Tanimizu and Mitaka, 2014). The cyotokine IL-6 may be involved in the transformation of hepatic progenitor cells into cancer stem cells. (Dang et al., 2014; He et al., 2013). In addition, Dragan et al. (1994) observed that the increase in BrdU labeling, indicating cell proliferation, occurred to a greater extent in altered hepatic foci (AHF).

There is no direct evidence addressing AFB1 induced critical gene mutations and the subsequent induction of AHF. However, in general the progression from chemical exposure to pre-neoplastic lesions appears to include six general mechanisms: 1) decrease in the activity of the Keap1/Nrf2/ARE pathway; 2) decrease in p53 function leading to increased survival in the presence of genomic instability; 3) changes in the tumor microenvironment within cells; 4) alterations in apoptosis; 5) changes in Wnt/β-catenin signaling, and 6) gene expression changes that may be related to cancer promotion/progression (Miyajima et al., 2014; Gong et al., 2013; Jia et al., 2013; Dang et al., 2014; He et al., 2013; Caballero et al., 2004; Ma and He, 2012; Gross-Steinmeyer and Eaton, 2012; Liby et al., 2008; Yates et al., 2007; Liby and Sporn, 2012; Ikeda et al., 2004; Haridas et al., 2004). In addition to these six possible mechanisms, epigenetic changes in DNA methylation may occur and may be related to the progression of cancer (Kwak et al., 2001; Honda et al., 2011; Shelton and Jaiswal, 2013; Fabregat, 2009; Zhang et al., 2003).

The following is an overview of these 6 general biological processes that appear to be involved in the progression of mutant cells (whether occurring as the result of direct chemical induction or increased due other events such as cell proliferation and clonal expansion of spontaneously occurring mutant cells) to AHF and pre-neoplastic lesions.

Decrease in p53 function A defective p53 gene may contribute to telomere dysfunction. Continuous hepatocyte renewal may be associated with telomere shortening and chromosomal instability [2-4]. In transgenic mice, telomere dysfunction enhanced tumor initiation but interfered with tumor progression/promotion. However, p53 mutation leading to inactivation of the gene, in combination with telomere dysfunction and increased hepatocyte turnover, may increase the risk of HCC (Zhang et al., 2006, 2012; Wu et al., 2013; Pogribny et al., 2011; Farazi et al., 2003).

The Tumor Microenvironment Intracellular signaling mechanisms are involved in the creation of tumor microenvironment. (Hanahan and Weinberg, 2011). These include alterations in cellular circuits governing proliferation, differentiation, motility, and viability. The x gene is one of the four genes in the Hepatitis B Virus (HBV) genome that is capable of trans-activating cellular genes to contribute to the development of a tumor microenvironment (Brechot et al., 2000, 2010). The x gene can be expressed through stable transfection into LE/6 cells, a cell line derived from hepatic oval cells harvested from rats fed a choline-deficient diet. When LE/6 cells that were transfected with HBx and injected into nude mice administered a single dose of 240 μg/kg AFB1 by IP injection, or in the drinking water continuously for 16 weeks, the mice developed HCC from these rat liver stem cells, indicative of the stem cell-origin of HCC and suggesting that HBV and x expression may foster a tumor microenvironment.

TGF-β has a dual effect on hepatocytes—inhibiting proliferation and inducing apoptosis. Experiments using TGF-β null mutant mice show that this signal is required for normal development and appears to be involved in destruction of damaged cells in the liver. TGF-β 1 also mediates interferon-induced apoptosis in AHF in rats. In rats, AFB1 altered biological processes in hepatic stem-like cells related to cell motion, cell adhesion, immune response, and signal transduction (Yang et al., 2014). Reduced levels of TGF-β produce an increase in E-cadherin and reduce cellular motility, one of the intracellular networks associated with the cancer hallmark of invasion and metastasis (Fransvea et al., 2008).

Effects on Apoptotic Signaling TGF-β normally inhibits growth and proliferation and induces apoptosis of damaged cells. In primary rat hepatocytes and a cell line (NRML) derived from these primary rat cells, TGF-β inhibited cell proliferation measured by DNA synthesis and the ability to form colonies. In vitro treatment of NRML cells with AFB1 blocked the TGF-β-mediated inhibition of cell growth and proliferation (McMahon et al., 1986). AFB1 transformed cells were also resistant to the growth-inhibiting effects of tumor necrosis factor-alpha (TNF-α) (Chapekar et al., 1989). However, BL9 cells, derived from rat hepatocytes transformed with reactive metabolites if activated AFB1, retained the sensitivity to the inhibitory effects of TGF-β on cell growth.

The product of the p53 gene mutated at codon 249 may itself impair the apoptotic process. In Hep3B human hepatoma cells, transfection with mutant p53 (p53mt249) increased transcription of insulin-like growth factor II (IGF-II). Wild-type p53 inhibited binding of the TATA-box binding protein (TBP) and the Sp1 transcription factors to the IGF-II promoter, possibly by binding to this promoter. However, p53mt249 increased the formation of transcription complexes, possibly due to lack of binding to the promoter. Chang liver cells show characteristics of hepatocytes; transfection with p53mt249 or addition of IGF-II blocked apoptosis mediated by TNF-α or the HBV x gene (Lee et al., 2000).

Adult tree shrews were administered AFB1 at 150 μg/kg/d five times weekly for 105 weeks. Interim sacrifices were performed every 30 weeks to observe liver histopathology, specifically immunohistochemical staining for p53, bcl-2, and bax, proteins in the p53 pathway that leads to apoptosis (Duan et al., 2005). Immunohistochemistry was also conducted to visualize survivin, a protein that acts directly on caspases to inhibit the activity of caspase-3 and caspase-7 to block the terminal pathway of apoptosis (Cheung et al., 2013). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect apoptotic cells.

Bcl-2 and bax were observed in the livers of less than 10% of treated animals at 30 weeks. This result seems somewhat paradoxical because these proteins are intermediates in the p53 apoptotic pathway; however, the determinant of apoptotic progression may be the bcl-2 / bax ratio (Korsmeyer et al., 1993). The actions of survivin occur much later in the apoptotic pathway and this protein was detected in 40% of livers. Also, the apoptotic index measured by TUNEL was increased significantly at 30 weeks (Duan et al., 2005).

Wnt/β-catenin Signaling Wnt/β-catenin signaling is a key determinant of liver cell proliferation and eventual differentiation to mature hepatocytes or cholangiocytes. This signaling cascade is generally inactive in healthy adult liver save for a small periportal region of a hepatic lobule. Hepatocytes in this region have constitutively active β-catenin that in turn regulates expression of genes associated with ammonia and xenobiotic metabolism (Monga, 2014). An increase in Wnt/β-catenin signaling appears to play a role in the development of hepatocellular cancers, and mutations in the β-catenin gene have been observed in HCCs from humans (Laurent-Puig and Zucman-Rossi, 2006; Devereux et al., 2001). In areas of high AFB1 exposure in China, β-catenin was overexpressed in a higher proportion of liver samples from HCC patients than in Malaysia where AFB1 exposure is lower (Chen Ban et al., 2004).

Paradoxically, AFB1 appears to increase microRNA-33a, which in turn down-regulates β-catenin (Fang et al., 2013). However, this study was conducted in human cancer cell lines and the signaling pathways in such cells may be considerably different than they are in vivo.

Genomic Changes in Response to AFB1 Administration of AFB1 to rat hepatic stem-like WB-F344 cells derived from oval cells produced changes in pathways related to TGF-β signaling, Wnt signaling, MAPK signaling, regulation of the cytoskeleton, cell adhesion, changes in energy metabolism, and ECM-receptor interaction (Yang et al., 2014). A number of these can be conceptually linked to the hallmarks of cancer (Hanahan and Weinberg, 2011). In another study, the AFB1-induced transcriptome, measured by RNA-Seq with increased resolution and sensitivity was compared to microarrays for pathways with differentially expressed genes, such as glutathione metabolism, cell cycle dysregulation, cancer pathways, Nrf2-mediated oxidative stress, and ten various intermediary and amino acid metabolism pathways (Merrick et al., 2013).

Empirical Support for Linkage (Qualitative Understanding)

In rats administered AFB1 then treated with oltipraz, decreases in the frequency of gamma-glutamyl transferase positive (GGT+) foci, the number of foci observed, and the size of the foci were all demonstrated, as well as decreased tumor incidence one to two years later (Roebuck et al., 1991). In rats treated with a single dose of 5 mg/kg AFB1, a progressive increase in the number and size of tigroid cell foci and neoplastic nodules was observed over a two-year period following dosing. Within these foci and nodules, the mitotic index was 100-fold higher than in the surrounding liver parenchyma (Bannasch et al., 1985).

Uncertainties or Inconsistencies

There is no direct information as to whether AFB1 induced mutations in critical genes leads to cellular proliferation and clonal expansion of mutant cells. It is known, however that the Hepatitis B virus plays a large role in the development of HCC in many endemic AFB1 regions, and viral infection is thought to interact with AFB1 exposure increasing the possibility that individual cells progress down the pathway to tumor formation. It seems reasonable that this infection may contribute to increased cell proliferation which could contribute to more clonal expansion (and, therefore, an increase in tumor formation) of AFB1-induced critical cancer gene mutant cells. It might also contribute to inflammation and increased cell proliferation which could result in an increased number of spontaneously mutant cells containing mutants in cancer critical genes (such as p53) thus also resulting in HCC (via a non-mutagenic MOA). This interaction between AFB1 and viral infection has motivated much research (Chittmittrapap et al., 2013; Farazi et al., 2006; Gouas et al., 2010; Kew 2003).

Quantitative Understanding of the Linkage

While studies have been conducted to quantitate clonal expansion and pre-neoplastic foci following exposure to AFB1, there are no studies that evaluate the quantitative relationship between cells carrying critical cancer gene mutations and clonal expansion/pre-neoplastic foci. This is primarily due to the lack of appropriate techniques. The chemoprevention data, however, indicate that the relationship between induced mutations in critical genes and AHF is likely not a linear one; additional data, such as with ACB-PCR techniques (Parsons et al., 2010), collected in a carefully designed study, could address this.

Evidence Supporting Taxonomic Applicability

In mammals, the identification of AHF as a pre-neoplastic lesion has been recognized for many years as part of the general etiology of HCC (Beer and Pitot, 1987; Dragan et al., 1995; Goldsworthy et al., 1986; Pitot et al., 1990; Sargent et al., 1989). In rats, the presence of AHF with altered growth characteristics has been observed in a number of studies (Bannasch et al., 1985; Fischer, 1986; Fischer et al., 1987; Gil et al., 1988; Godlewski et al., 1985; Manson et al., 1984; Newberne, 1976; Nishizumi et al., 1977; Roebuck et al., 1991; Youngman and Campbell, 1992). The mechanisms involved in the formation of AHF appear to be generalizable across animals and likely apply to fish and birds as well as mammals.

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